Abstract
Growth hormone (GH) is an important mitogenic stimulus for the insulin-producing beta-cell. We investigated the effects of GH on Ca(2+) handling and diacylglycerol (DAG) and cAMP formation in the beta-cell. GH elicited a rapid increase in the cytoplasmic free [Ca(2+)], which required extracellular Ca(2+) and was also blocked by pertussis toxin or protein kinase C (PKC) inhibition. GH also elevated islet DAG content, which should lead to PKC activation. Pertussis toxin and PKC inhibitors obliterated the mitogenicity of GH, suggesting involvement of GTP-binding proteins. PKC activation stimulated beta-cell proliferation, and it also activated phospholipase D. Islet cAMP content was not elevated by GH. Addition of a specific protein kinase A antagonist failed to influence the mitogenicity of GH, whereas a stimulatory cAMP agonist stimulated beta-cell replication. We conclude that GH rapidly increases the beta-cell cytoplasmic free [Ca(2+)] and also evokes a similar increase in DAG content via a phosphatidylcholine-specific phospholipase C, but does not affect mitogen-activated protein kinases, phospholipase D, or the cAMP signaling pathway. This rise in DAG may be of importance in translation of the stimulatory signal of GH into a proliferative response by the beta-cell, which seems to occur through GTP-binding proteins and PKC-dependent mechanisms.
Highlights
Long-term alterations in pancreatic islet -cell mass constitute an important means to accommodate an increased demand for insulin
In this study, we have utilized an in vitro system of fetal rat islets enriched in rapidly proliferating -cells to study the putative role of [Ca2ϩ]i, GTP-binding proteins, polyphosphoinositide and phosphatidylcholine hydrolysis, and protein kinase activation in the proliferative signal induced by growth hormone (GH)
The rise in [Ca2ϩ]i evoked by GH was found to be due to influx of Ca2ϩ across the plasma membrane since it did not occur in the absence of extracellular Ca2ϩ (Fig. 2C)
Summary
Materials—Recombinant human GH was kindly provided by Dr Anna Skottner (Pharmacia & Upjohn Corp., Stockholm, Sweden). Monolayers of cells were prepared as described [15] by shaking in a Ca2ϩ-free medium containing EGTA and cultured overnight on plastic coverslips. CAMP Measurements—For cAMP measurements, islets in groups of 50 that had been cultured for 3 days in the presence or absence of GH or the desired test substances were quickly rinsed once in ice-cold PBS and swiftly transferred to Eppendorf tubes containing 150 l of ice-cold 6% trichloroacetic acid. These tubes were immediately sealed, plunged into liquid nitrogen, and stored at Ϫ80 °C, pending analysis. Means Ϯ S.E. were calculated, and groups of data were compared using Student’s paired or unpaired t test
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